The focus of this proposal is to develop a novel "conditioning" approach that will replace myelotoxic agents to establish chimerism in NOD mice. We will induce immune deviation to promote host-versus-graft hyporesponsiveness, thereby giving the hematopoietic stem cell (HSC) an opportunity to engraft and establish subsequent self-perpetuating deletional tolerance to islet allografts. Our recent studies in a mouse model suggest that the primary role for conditioning for HSC transplantation is to suppress host-versus-graft alloreactivity, rather than to prepare vacant niches in the recipient's bone marrow compartment. This observation suggests that one could replace myelotoxic agents with antigen-specific approaches to induce host-versus-graft hyporeactivity or anergy at the time of HSC transplantation. As the mechanisms underlying T cell activation are defined, highly specific approaches to suppress this alloreactivity have emerged. In AIM I. we will ESTABLISH CHIMERISM THROUGH IMMUNE DEVIATION OF THE RECIPIENT. We will immunomodulate the recipient: (a) targeting alloreactive cells in the host microenvironment;(b) inducing anergy and/or antigen-specific apoptosis of alloreactive host cells;and (c) through generation of regulatory T cells (Treg), and develop a novel nonmyeloablative conditioning regimen to induce antigen-specific hyporesponsiveness to the HSC and islet allografts. Cell-based therapies have great potential for inducing transplantation tolerance. Of greatest interest are the new subpopulations of bone marrow-derived dendritic cells (DC) that have recently been shown to be potently tolerogenic in vitro under certain circumstances. We are the first to demonstrate an in vivo engraftment-enhancing effect for precursor plasmacytoid DC (p-preDC). The exploitation of this discovery in vivo and its potential to reduce the need for myelotoxic conditioning has not yet been tested. Hematopoietic growth factors have also been used to drive the immune response to a tolerogenic T helper 2 (Th2) phenotype through production of p-preDC or other tolerance-promoting cells (graft facilitating cells {FC}) that in turn generate Treg. In AIM II, we will USE PRE-TRANSPLANT IMMUNOMODULATION OF THE DONOR WITH HEMATOPOIETIC GROWTH FACTORS TO GENERATE TOLEROGENIC CELLS IN THE HSC ALLOGRAFT. We will use these factors and the cells they generate to modulate the tolerogenicity of the donor marrow inoculum in vivo to tip the immune milieu in favor of graft acceptance, enhancing bone marrow chimerism without myelotoxic conditioning. We will examine the mechanism by which this occurs and identify which cell types in the graft are critical to tolerance induction. P-preDC exposed to apoptotic donor antigens are potently tolerizing in vitro through generation of Treg. The therapeutic application of this approach has not been tested in vivo. In AIM III, we will USE EX VIVO IMMUNOMODULATION OF THE MARROW to expand p-preDC and FC and induce a tolerogenic inoculum for HSC transplantation.